The MALDI-TOF MS determination of yeast proteins producing $H_2S$

MALDI-TOF MS를 이용한 효모에서의 황화수소 생성 단백질의 동정

  • Cho, Hyun-Nam (Dept. of Applied Chemistry, Kumoh National Institute of Technology) ;
  • Fan, Lu-An (Dept. of Applied Chemistry, Kumoh National Institute of Technology) ;
  • Yoo, Dong-Chan (Dept. of Applied Chemistry, Kumoh National Institute of Technology) ;
  • Yang, Seun-Ah (The Center for Traditional Microorganism Resources, Keimyung University) ;
  • Lee, In-Seon (The Center for Traditional Microorganism Resources, Keimyung University) ;
  • Kim, Jae-Hyung (Bio Institute, Dong-il SHIMADZU Corp.) ;
  • Baek, Hyo-Hyun (Bio Institute, Dong-il SHIMADZU Corp.) ;
  • Jhee, Kwang-Hwan (Dept. of Applied Chemistry, Kumoh National Institute of Technology)
  • 조현남 (금오공과대학교 자연과학부 응용화학) ;
  • 판루안 (금오공과대학교 자연과학부 응용화학) ;
  • 유동찬 (금오공과대학교 자연과학부 응용화학) ;
  • 양선아 (계명대학교 전통미생물자원개발 및 산업화연구센터) ;
  • 이인선 (계명대학교 전통미생물자원개발 및 산업화연구센터) ;
  • 김재형 (동일시마즈(주)) ;
  • 백효현 (동일시마즈(주)) ;
  • 지광환 (금오공과대학교 자연과학부 응용화학)
  • Published : 2008.10.31


Hydrogen sulfide ($H_2S$) is a by-product of metabolism of amino acids including sulfur and alcoholic fermentation, it is generally thought of in terms of a poisonous gas. Though $H_2S$ can have a negative impact on the perceived quality of fermented drinks due to an undesirable aroma, it plays prominent roles as a neuromodulator in the mammalian brain as well as a smooth muscle relaxant. Nowadays studies on the proteins which produce $H_2S$ are carried out in various fields such as structure, function, and metabolism. Here we propose to develop a simple and rapid $H_2S$ forming assay method, which will lead to speed up preparing the $H_2S$ forming proteins for identification by MALDI-TOF MS analysis. We detected three kinds of proteins which produce $H_2S$ in the crude extract of Saccharomyces cerevisiae. Those proteins were cystathionie $\beta$-synthase, O-acetylserine sulfhydrylase, and cystathionine $\gamma$-lyase.

생체에서의 황화수소는 의약 분야와 발효 산업에 있어서 중요한 역할을 하고 있다. 본 연구에서는 Saccharomyces cerevisiae을 이용하여 기질인 L-cysteine과 $\beta$-mercaptoethanol로부터 $\beta$-replacement 반응에 인한 황화수소를 발생시킬 수 있는 효소를 간편하고 신속하게 동정하는 방법의 확립을 목적으로 하였다. 효소에 의해 발생된 황화수소와 Pb-acetate의 반응으로 생성된 Pb-S를 gel상에서 간편하게 확인한 후, 확인된 단백질들을 이온교환컬럼를 수행한 후 gel에서 추출하는 방법으로 MALDI-TOFMS의 시료를 간단히 얻을 수 있었다. PMF 방법과 MS/MS ion search 분석을 통해 간편하게 효모에서 황화수소를 형성할 가능성이 있는 세가지 단백질의 동정에 성공하였다. 이 세 가지 단백질은 CYS3, CYS4, MET17 유전자의 단백질로서 cystathionine $\gamma$-lyase, CBS, OASS 임이 밝혀졌다. 그리고 이 세 단백질들은 L-cysteine과 $\beta$-mercaptoethanol의 존재하에서 황화수소를 실제로 생성함을 확인하였다. 본 연구에서 표적의 물질을 생산하는 단백질들을 젤상에서의 활성측정과 MALDI-TOF MS를 이용하여 간단히 그리고 정확하게 동정하는 방법을 확립하였다.



  1. Kimura, H. (2002), Hydrogen sulfide as a neuromodulator, Mol. Neurobiol. 26(1), 13-19
  2. Eto, K., T. Asada, K. Arima, T. Makifuchi, and H. Kimura (2002), Brain hydrogen sulfide is severely decreased in Alzheimer's disease, Biochem. Biophys. Res. Commun. 293(5), 1485-1488
  3. Eto, K. and H. Kimura (2002), The production of hydrogen sulfide is regulated by testosterone and S-adenosyl-L-methionine in mouse brain, J. Neurochem. 83(1), 80-86
  4. Chen, X., K. H. Jhee, and W. D. Kruger (2004), Production of the neuromodulator $H_2B$ by cystathionine ${\beta}$-synthase via the condensation of cysteine and homocysteine, J. Biol. Chem. 279(50), 52082-52086
  5. Jhee, K. H., H. N. Cho, S. A. Yang, and I. S. Lee (2007), Biochemical characteristics for the cofactor free mutant of yeast homocysteine catalyzing enzyme, cystathionine ${\beta}$-synthase, Kor. J. Microbiol. Biotechnol. 35(3), 196-202
  6. Shan, X. and W. D. Kruger (1998), Correction of disease-causing CBS mutation in yeast, Nat. Genet. 19, 91-93
  7. Spiropoulos, A. and L. F. Bisson (2000), MET17 and hydrogen sulfide formation in Saccharomyces cerevisiae, Appl. Environ. Microbiol. 66(10), 4421-4426
  8. Mendes-Ferreira, A., A. Mendes-Faia, and C. Leao (2004), Growth and fermentation patterns of Saccharomyces cerevisiae under different ammonium concentrations and its implications in winemaking industry, J. Applied Microbiology 97, 540-545
  9. Matthews, A., A. Grimaldi, M. Walker, E. Bartowsky, P. Grbin, and V. Jiranek (2004), Lactic acid bacteria as a potential source of enzymes for use in vinification, Appl. Environ. Microbiol. 70(10), 5715-5731
  10. Edwards, C. G. and J. C. Bohlscheid (2007), Impact of pantothenic acid addition on $H_2S$ production by Saccharomyces under fermentative conditions, Enzyme and Microbial Technology 41, 1-4
  11. Linderholm, A. L., C. L. Findleton, G. Kumar, Y. Hong, and L. F. Bisson (2008), Identification of genes affecting hydrogen sulfide formation in Saccharomyces cerevisiae, Appl. Environ. Microbiol. 74(5), 1418-1427
  12. Hansen, R., S. Y. Pearson, J. M. Brosnan, P. G. Meaden, and D. J. Jamieson (2006), Proteomic analysis of a distilling strain of Saccharomyces cerevisiae during industrial grain fermentation, Appl. Biochem. Biotech. 72, 116-125
  13. Trabalzini, L., A. Paffetti, A. Scaloni, F. Talamo, E. Ferro, G. Coratza, L. Bovalini, P. Lusini, P. Martelli, and A. Santucci (2003), Proteomic response to physiological fermentation stresses in a wild-type wine strain of Saccharomyces cerevisiae, Biochem. J. 370, 35-46
  14. Linderholm, A. L., T. L. Olineka, Y. Hong, and L. F. Bisson (2006), Allele diversity among genes of the sulfate reduction pathway in wine strains of Saccharomyces cerevisiae, Am. J. Enol. Vitic. 57(4), 431-440
  15. Ono, B. I., T. Hazu, S. Yoshida, T. Kawato, S. Shinoda, J. Brzvwczy, and A. Paszewski (1999), Cysteine biosynthesis in Saccharomyces cerevisiae: a new outlook on pathway and regulation, Yeast 15(13), 1365-1375<1365::AID-YEA468>3.0.CO;2-U
  16. Swiegers, J. H. and I. S. Pretorius (2007), Modulation of volatile sulfur compounds by wine yeast, Appl. Microbiol. Biotechnol. 74, 954-960
  17. D'Andrea, R., Y. Surdin-Kerjan, G. Pure, and H. Cherest (1987), Molecular genetics of met17 and met25 mutants of Saccharomyces cerevisiae: intragenic complementation between mutations of a single structural gene, Mol. Gen. Genet. 207, 165-170
  18. Jhee, K. H., P. McPhie, and E. W. Miles (2000), Domain architecture of the heme-independent yeast cystathionine ${\beta}$-synthase provides insights into mechanisms of catalysis and regulation, Biochemistry 39(34), 10548-10556
  19. Kruger, W. D. and D. R. Cox (1994), A yeast system for expression of human cystathionine ${\beta}$ -synthase: structural and functional conservation of the human and yeast genes, Proc. Natl. Acad. Sci. 91, 6614-6618
  20. Bradford, M. M. (1976), A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding, Analytical Biochemistry 72, 248-254
  21. Muunchbach, M., M. Quadroni, G. Miotto, and P. James (2000), Quantitation and facilitated de novo sequencing of proteins by isotopic N-terminal labeling of peptides with a fragmentation-directing moiety, Anal. Chem. 72, 4047-4057
  22. Yamagata S. (1976), O-Acetylserine and O-acetylhomoserine sulfhydrylase of yeast. Subunit structure, J. Biochem. 80(4), 787-797
  23. Strambini, G. B., P. Cioni, and P. F. Cook (1996), Tryptophan luminescence as a probe of enzyme conformation along the O-acetylserine sulfhydrylase reaction pathway, Biochemistry 35(25), 8392-8400
  24. Szabo, C. (2007), Hydrogen sulphide and its therapeutic potential, Nat. Rev. Drug. Discov. 6(11), 917-935